The Effect Of Bone Morphogenetic Protein-4 On Follicles From Cryopreserved Human Ovarian Tissue By Vitrification

With the development of medical technology, survival rates of children malignant tumors who were treated by chemical medicine or radiology have been improved dramatically. While many treatments administered for such women may carry a substantial risk for menstrual disorder ,premature ovarian failure, and even loss of reproductive activity, which may severely affect patients'fertility and quality of life. At the same time, because of the increasing social competitive pressure, many women have to postpone the birth time, which may lead to the decrease of ovarian reserve and may be harmful to the offspring. So the protection of female reproductive resources especially for patients receiving anti-tumor therapy and removing the fear of disturbance in the rear of occupational women, becomes the hot topic of reproductive medicine.At present, there are many methods for female reproductive protection, for example, oocyte freezing, transposition by surgery, drugs and ovarian tissue freezing. Comparing to some other methods for female reproductive protection, ovarian tissue freezing in human can better restore a lot of primordial follicles, Because primordial follicles have several characteristics that make them less vulnerable to cryoinjury than embryo and mature follicles, is suitable for children, adolescences and fertility women, which does not rely on menstrual cycle, does not need superovulation , and does not delay patients'treatments. According to data, People have not only restore natural menstruation but also get pregnancy and delivery by IVF, after ovarian tissue freezing and transplantation. Ovarian tissue freezing is believed to be a promising ovarian function protection method.Slow cooling-rapid thawing was the mainly method in the past research. But slow cooling needs artificial seeding and expensive equipments, and its operation is relatively complicated and expends more liquid nitrogen. Because ovarian cortex is very thick and has many types of cells, which need different rates of cooling and different time to seed,slow cooling protocol is a little easy to form ice crystals and causes solute damage because follicles expose to high concentration of both intra- and extracellular electrolytes for a long time. These can reduce survival rate of follicles. Now, there is a new cryopreservation protocol named vitrification. Vitrification is based on using high concentrations of cryoprotectants to solidify the cell in a glass state and mostly reduce ice formation .Its operation is very simple and can be finished in a short time without expensive equipments, which greatly lessens solute damage. Healthy babies were born following vitrification of human oocytes, embryos and blastocysts. But the data on vitrification of human ovarian tissue are limited. Therefore, vitrification technique becomes new hot topic for the protection of ovarian function protection. The aim of this investigation is to evaluate the survival vigor of cultured frozen-thawed human ovarian tissue.During the follicle development, the transition of primordial to primary and the transition of primary to secondary are believed as the key steps of follicle growth. But, the transition mechanism of primordial to primary follicle is still unclear, the precise substance and mechanism initiating primordial follicle growth needs further research. Recently, growth factor attracts more attention to explore the transition mechanism and decrease the atretic rate of follicle during the in vitro culture, especially the members of ovarian autoregulatory system. From recent accumulating in vivo and in vitro evidence, it appears that members of the bone morphogenetic protein(BMP)family of cytokines and their receptors are strongly implicated in ovarian function, controlling folliculogenesis and ovulation rate. Of all the growth factors in the ovary, the members of the transforming growth factor-β(TGF-β)superfamily figure most prominently in the regulatory events of folliculogenesis, BMPs comprise one of the largest subgroups of ligands in the TGF-βsuperfamily, with 15 BMPs having been described to date. BMP-4 was first characterized for its role in bone and cartilage metabolism. during development, the function of BMP-4 in mammal ovary was reported by Shimasaki in 1999. In rat ovaries, high levels of BMP-4 were expressed in theca cells while BMP-4 receptor mRNAs for BMPR-Ⅱ,BMPR-ⅠA, and BMPR-ⅠB have been detected in both oocytes and granulose cells. In vitro culture of neonatal rat ovaries, exogenous BMP-4 was found to promote the primordial-to-primary follicle transition, and decrease the apoptosis rates. In rat experiment, it was found that physiological concentrations of BMP-4 enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively, it was hypothesized that BMP-4 might be the long-sought "luteinization inhibitor". The inhibition of progesterone production on its own in the absence of FSH as well as in the FSH-induced condition was investigated in vitro culture of ovine granular cells(GCs). suggesting that BMP-4 may play important role in follicle growth, development and survival.The aim of this research was to study the effect of human ovarian tissue cryopreservation by vitrification ,then after frozen-thawed, to study the actions of BMP-4 on human primordial-to-primary follicle transition and further development, to observe the influence of BMP-4 on the basal estradiol and progesterone production of follicles, and to investigate the effect of BMP-4 on the survival of follicles and stroma cells, to provide theoretic and experimental basis for the further exploration of the primordial-to-primary transition mechanism and the clinical application of follicle in vitro culture and cryopreservation technology.Materials and Methods:1. Source of the ovariesOvarian cortex tissues were obtained with informed consent by ovarian biopsy from 32 patients who undergone ovariectomy from April to November in 2008 in the Department of Gynecology and Obstetrics of Guang Zhou First Municipal People,s Hospital, the mean age was 29.66±4.25 years(16-37 years).The patients had normal menstrual cycles without hormonal administration, no endocrine disturbance, and the diameter of ovarian cyst was less than 7cm.2. Experimental protocol2.1 Human tissue cryopreservation and cultureOvarian tissue biopsied either by laparoscopy or laparotomy without prominent follicles or luteal corpus was collected into HEPES-buffered minimum essential medium (MEM), transported to the laboratory within 30 minutes, rinsed in HEPES-buffered MEM twice under room temperature and placed in the culture dish with this medium. The ovaries were stripped of surrounding tissue, after which the medulla was removed ,then the tissue was cut into pieces of approximately 2mm×2mm×1mm with a scalpel and mounted needles, under a stereo microscope. Tissue pieces from the same patient were randomly distributed to four groups: fresh group(A); other groups(B,C and D) were cryopreserved by vitrification: non-culture group(B),α-MEM culture group(C) and BMP-4 culture group(D). The tissue pieces of group C and group D were incubated at 37℃under 5% CO2 for 15 days, every second day about 400μl of the culture medium was replaced by fresh medium, and the replaced medium was collected and stored -20℃to measure the concentration of estradiol and progesterone.2.2 Histological analysisThe tissue pieces of the fresh, non-culture group, theα-MEM culture group and the BMP-4 culture group by vitrification after culture finished were under histological analysis, serially sectioned at 5μm, stained with haematoxylin and eosin and analyzed under microscopy (×400). The number of primordial, primary and secondary follicles of 10 high power field (×400)was counted to calculate the percentage of primordial and developing (including primary and secondary) follicles in the three groups.2.3 Hormone level measurementAfter the ovarian tissue culture, the changed medium stored in EP tube was thawed to measure the concentration of estradiol and progesterone by Automated Chemiluminescence System (ADVIA Centaur).Results1.The effecf of cryopreservation on the percentage of follicleIn the frozen fragments,the percentage of both primordial and primary follicles respectively was not different compared with the ease of fresh human ovarian fragments(P>0.05).2.Morphology evaluation of follicles from cryopreserved human ovarian tissue by vitrificationThe percentage of morphologically normal both primordial and primary follicles was similar between the non-cryopreserved and cryopreserved human ovarian tissue(P>0.05).3. The effect of BMP-4 on follicle developmentAfter 15 days culture , 53.29±18.26% and 43.38±22.59% of follicles were in primordial stages in the group C and group D, the percentage of primordial follicles in the non-culture group which was 84.63±8.01% was significantly higher than in group C and group D, P<0.01,while there was no difference between group C and D(P>0.05).The percentage of developing follicles in the group C and group D was 46.71±18.26%and 56.62±22.59%respectively, with no statistical significance, but markedly higher than in the non-culture group, the developing follicle percentage of which was 15.37±8.01%,P<0.01.4. The effect of BMP-4 on basal estrodiol and progesterone production .The concentration of estrodiol in group C and group D was 1011.13±321.50ng/L and 1186.17±405.78ng/L respectively, It was a little bit more in group D,but with no statistical significance. However, the concentration of progesterone in group D was 4.61±2.69ug/L, significantly lower than in group C, which was 7.02±3.09ug/L,P<0.05.Conclusions1. There are not only a lot of primordial follicles but also many primary follicles which can be well preserved in human ovarian tissue cryopreservation.2. Through histological analysis, vitrification can preserve morphology of primordial and primary follicles well, is a favorable way in human ovarian cryopreservation.3. BMP-4 could help to promote the primordial-to-primary vitrified follicle transition of human ovarian tissue in vitro culture.4. BMP-4 could significantly inhibit the basal progesterone secretion and may promote estrodiol secretion in vitro culture of human granular cells.